Targeting the PI3K pathway in cancer: are we making headway?

The phosphoinositide 3-kinase (PI3K) pathway is one of the most commonly activated pathways in human cancer and has roles in cell proliferation, apoptosis, protein synthesis, and metabolism. The PI3K pathway can be activated by amplification or activating mutation of upstream receptor tyrosine kinases, and by mutations or deletions downstream in the pathway. Trastuzumab, a monoclonal antibody targeting the human epidermal growth factor receptor 2 (HER2), has been one of the most successful and most widely used targeted therapies. However, many HER2-positive cancers are not sensitive to HER2-based therapies or become resistant during treatment; downstream activation of the pathway is one of the causes of resistance. Because of the common activation of the PI3K pathway in cancer, compounds targeting proteins downstream in the pathway have been developed in recent years. The mammalian target of rapamycin (mTOR) inhibitors everolimus and temsirolimus have been shown to be beneficial in certain cancer types; many other inhibitors of the PI3K pathway are in various stages of clinical development. Ongoing research should clarify which molecular cancer subtypes are most susceptible to specific compounds and explore combinatorial approaches, ultimately leading to individualized patient treatment.

Chronic myeloid leukemia (CML), a myeloproliferative hematopoietic cancer, is caused by a genetic translocation between chromosomes 9 and 22. This translocation produces a small Philadelphia chromosome, which contains the Bcr-Abl oncogene. The Bcr-Abl oncogene encodes the BCR-ABL protein, upregulates various signaling pathways (JAK-STAT, MAPK/ERK, and PI3K/Akt/mTOR), and out of which the specifically highly active pathway is the PI3K/Akt/mTOR pathway. Among early treatments for CML, tyrosine kinase inhibitors (TKIs) were found to be the most effective, but drug resistance against kinase inhibitors led to the discovery of novel alternative therapies. At this point, the PI3K/Akt/mTOR pathway components became new targets due to stimulation of this pathway in TKIs-resistant CML patients. The current review article deals with reviewing the scientific literature on the PI3K/Akt/mTOR pathway inhibitors listed in the National Cancer Institute (NCI) drug dictionary and proved effective against multiple cancers. And out of those enlisted inhibitors, the US FDA has also approved some PI3K inhibitors (Idelalisib, Copanlisib, and Duvelisib) and mTOR inhibitors (Everolimus, Sirolimus, and Temsirolimus) for cancer therapy. So far, several inhibitors have been tested, and further investigations are still ongoing. Even in Imatinib, Nilotinib, and Ponatinib-resistant CML cells, a dual PI3K/mTOR inhibitor, BEZ235, showed antiproliferative activity. Therefore, by considering the literature data of these reviews and further examining some of the reported inhibitors, which proved effective against the PI3K/Akt/mTOR signaling pathway in multiple cancers, may improve the therapeutic approaches towards TKI-resistant CML cells where the respective signaling pathway gets upregulated.

Aberrant activation of the PI3K/Akt pathway is found in approximately 70% of breast cancers. We recently identified a recurrent rearrangement that produces an in-frame fusion protein containing Akt3 and the scaffolding protein MAGI3. MAGI3-Akt3 is the first fusion protein that has been identified in the PI3K pathway. We propose that MAGI3-Akt3 is a novel oncogene in breast cancer and that the oncogenic activity of the fusion protein results from disruption of the PI3K/Akt pathway. Within the fusion, Akt3 has a disrupted PH domain prior to glutamate 17 (E17). As a result the fusion protein is constitutively phosphorylated in the Akt3 kinase domain and expression of MAGI3-Akt3 in breast cancer cells enhances Akt substrate phosphorylation. Constitutive MAGI3-Akt3 kinase activity can be inhibited by ATP-competitive small molecule Akt inhibitors but is resistant to the allosteric Akt inhibitor MK2206 which is currently in phase II clinical trials. Our results indicate that ATP competitive Akt inhibitors should be evaluated in clinical trials for the treatment of fusion-positive breast cancers. The fusion protein also contains a severely truncated portion of the scaffolding protein MAGI3. In particular, the fusion lacks the second PDZ domain of MAGI3 that has been reported to bind to PTEN and be required for the inhibitory effect of PTEN on PI3K pathway activity. We have demonstrated that disruption of MAGI3 in breast cancer cell lines does indeed promote PI3K pathway hyperactivity in a PTEN-dependent manner. In addition, the fusion lacks the fifth PDZ domain of MAGI3 and our studies indicate that this disrupts the interaction of MAGI3 with PHLPP2, a phosphatase that directly dephosphorylates Akt at pS473. Our studies therefore demonstrate that the MAGI3-Akt3 translocation significantly perturbs PI3K pathway activity via activation of an oncogene (Akt3) and dysregulation of two tumor suppressors (PTEN and PHLPP2). We are currently continuing studies to elucidate the oncogenic mechanism(s) of MAGI3-Akt3 in breast cancer. Citation Information: Mol Cancer Ther 2013;12(11 Suppl):B195. Citation Format: Kristin Brown, Alex Toker. MAGI3-Akt3, a novel fusion protein in the PI3K pathway in cancer. [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2013 Oct 19-23; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2013;12(11 Suppl):Abstract nr B195.

All in the family: Clueing into the link between metabolic syndrome and hematologic malignancies

Background: Inhibitors of the mammalian target of rapamycin (mTOR) pathway including everolimus and temsirolimus have been used extensively in cancer patients. Their use is associated with stomatitis, an adverse event resulting in morbidity and treatment interruptions or discontinuation. This study was conducted to determine the overall incidence and risk of stomatitis in cancer patients treated with the mTOR inhibitors by a meta-analysis of randomized controlled clinical trials (RCTs). Patients and Methods: Databases from PubMed and abstracts presented at the American Society of Clinical Oncology annual meetings up to October 2013 were searched for relevant studies. Eligible studies included RCTs using everolimus and temsirolimus at approved doses in cancer patients. Summary incidences, relative risks (RR), and 95% confidence intervals (CI) were calculated using a random- or fixed-effects model depending on the heterogeneity of the included trials. Results: A total of 11 RCTs with 4,752 patients (mTORs: 2,725, controls: 2,027) with a variety of solid tumors were included in the analysis. The incidences of all-grade (grade 1–4) and high-grade stomatitis (grade 3–4) were 33.5% (95% CI: 21.9–47.6%) and 4.1% (95% CI: 2.6–6.3%), respectively. The incidence of high-grade stomatitis significantly varied with tumor types (p = .004), and mTOR inhibitors (temsirolimus vs. everolimus, p < .001). In comparison with controls, mTOR inhibitors significantly increased the risk for developing all-grade stomatitis (RR: 4.04, 95% CI: 3.13–5.22, p < .001) and high-grade stomatitis (RR: 8.84, 95% CI: 4.07–19.22, p < .001). Conclusions: The mTOR inhibitors everolimus and temsirolimus significantly increased the risk of high-grade stomatitis in cancer patients. Efforts towards the prevention, treatment, and identification of individuals at risk may allow for improved quality of life and consistent dosing.

BackgroundTwo novel mammalian targets of rapamycin (mTOR) inhibitors everolimus and temsirolimus are now approved by regulatory agencies and have been widely investigated among various types of solid tumors, but the risk of fatal adverse events (FAEs) with these drugs is not well defined.MethodsWe searched PubMed, EMBASE, and Cochrane library databases for relevant trials. Eligible studies included prospective phase II and III trials evaluating everolimus and temsirolimus in patients with all malignancies and data on FAEs were available. Statistical analyses were conducted to calculate the summary incidence, RRs and 95% confidence intervals (CIs) by using either random effects or fixed effect models according to the heterogeneity of the included studies.ResultsA total of 3322 patients with various advanced solid tumors from 12 trials were included. The overall incidence of mTOR inhibitors associated FAEs was 1.8% (95%CI: 1.3–2.5%), and the incidences of everolimus related FAEs were comparable to that of temsirolimus (1.7% versus 1.8%). Compared with the controls, the use of mTOR inhibitors was associated with an increased risk of FAEs, with a RR of 3.24 (95%CI: 1.21–8.67, p = 0.019). On subgroup analysis, a non-statistically significant increase in the risk of FAEs was found according to different mTOR inhibitors, tumor types or controlled therapy. No evidence of publication bias was observed.ConclusionWith the present evidence, the use of mTOR inhibitors seems to increase the risk of FAEs in patients with advanced solid tumors. More high quality trials are still needed to investigate this association.

By analyzing the gene expression of endometrial carcinoma (EC) patients, the key factors in PI3K signaling pathway and its related genes mediating EC were explored. The EC samples and normal endometrial samples were downloaded from TCGA database and GTEx database. The R language "limma" package was used for differential analysis, and the expression level of genes in each tissue was analyzed by "gganatogram" package. Functional enrichment analysis of differential genes was carried out by KOBAS, an online bioinformatics website. The correlation between key genes and differential genes was evaluated using TCGA data and GTEx combined gene expression data. The corresponding clinical data were downloaded from TCGA database and GTEx database, and the R language "survival" package was used to assess the potential of candidate differential genes as a key factor of EC. Based on the combined differential analysis of TCGA and GTEx databases, 299 genes with significant differential in expression were finally got. Functional enrichment analysis revealed that genes were predominantly enriched in the entry of "Pathways in cancer", including RAC2 and PIK3R3 genes which were related with the abnormal PI3K pathway in cancer. PIK3R3, a key gene in the PI3K signaling pathway, was highly-expressed in EC. SPDEF, GCNT2, KIAA1324, C9orf152, MARVELD3, and APEX2 genes were found to be positively correlated with PIK3R3 in EC, all of which were highly expressed in EC. KM survival analysis showed that SPDEF, GCNT2, KIAA1324 and C9orf152 were significantly correlated with patients' survival. ROC analysis showed that SPDEF, GCNT2, KIAA1324 and C9orf152 gene could be used as potential markers for prognosis and survival of EC patients. It was found that PIK3R3, a key gene in the PI3K signaling pathway, was highly expressed in EC. The SPDEF, GCNT2, KIAA1324 and C9orf152 genes were also highly expressed in EC, and were positively correlated with PIK3R3 in EC. Moreover, they are significantly correlated with the patients' survival, suggesting that they may be potential markers for the prognosis of patients with EC.

SY01-02: Targeting stem cell pathways in human cancer

The PI3K/AKT/mTOR pathway is one of the most frequently altered pathways in human cancers. Although the loss of function of PTEN in prostate cancer increases the activity of the PI3K/AKT/mTOR pathway, clinical studies thus far have been disappointing. Studies of the mammalian target of rapamycin (mTOR) inhibitors rapamycin, everolimus, and temsirolimus when used as single-agents failed to demonstrate significant clinical activity in metastatic castration-resistant prostate cancer (CRPC) patients. Novel inhibitors of this pathway such as dual inhibitors of the phosphatidylinositol 3-kinase (PI3K) and mTOR pathway are now in clinical development. The most promising future use of these agents will likely be in combination with other agents such as potent AR pathway inhibitors.

Rapamycin selectively inhibits expression of an inducible keratin (K6a) in human keratinocytes and improves symptoms in pachyonychia congenita patients

Background: Mutations guide targeted therapy in the personalized medicine. In the opening chapter of our recently edited book (Dey et al., 2016), Prof. L. Cantley elegantly elucidated the basic signaling of the PI3K pathway in cancers. Mutations in the PI3K pathway are not only common and subtype-specific in BC but are also contextual. Alterations in DNA damage repair (DDR) pathway involving HRD (Homologous Recombination Defect) genes are one of the important contextual events of the upregulation of the PI3K pathway (De et al., 2016). Aim: Here we interrogated the contextuality of alterations of the PI3K and DDR pathway genes in our Avera patients. The mechanism of contextual cooperation between the pathwayswas experimentally validated. Methods: We examined mutation profile (FoundationOne) of our patients (Avera Cancer Institute) and patients fromthe TCGA data (cBioPortal). We validated the cooperation of the two pathwaysexperimentally by the synergy model of mutation-specific drugs; PI3K-PTEN-mTOR pathway inhibitor(s) and PARP inhibitor(s) using TNBC model.Results: We analyzed alterations of 17 and 12 genes of the PI3K and DDR pathways respectively in subtypes of BC. In luminal A and HER2-enriched (TCGA, Nature 2012), the alteration of PIK3CA reached 49 % and 47% as compared to 37 % in luminal B and 25% in basal-like.In the basal-like/TNBC subtype (cBioPortal) 12 DDR pathway genes (CHEK1/2, RAD51, BRCA1/2, MLH1, MSH2, ATM, ATR, MDC1, PARP1, FANCF) were altered in 90.1 % of cases, and 17 PI3K pathway genes were altered in 88.9 % of cases.Our ER+ve patients presented a diverse variety of PIK3CA mutations (E545K, E545A, E545G, E542K, E453K, E762K, E365K, N345K, C420R, E81K, Q546R, C420R, E726K, E81K, E970K, H1047R, H1047L P104L, P539R, G106R,G1049R, R93Q,N345T, V105_E109&amp;gt;E, L113del, K111del) as compared to a less diverse type of PIK3CA mutations (Amplification, E542K, H1047R) in our TNBC patients. In our TNBC patients, the predominant type of mutation in PI3K pathway genes was found in PTEN consisting of Y68C, Y180*, loss, loss exons 1-5, and deletion exon1. The other most common mutation found in TNBC patients was in TP53 (&amp;gt;80%) and somatic BRCA1/2 (˜15%) genes. The interaction between the two pathways was evaluated using the mostly altered oncogenes and tumor suppressor genes (PTEN, AKT1/2, TSC1/2, mTOR, RICTOR, RHEB, BRCA1/2, ATM, ATR, FANCF) applying STRING10 to test the association at the highest 0.900 confidence views. Finally, we experimentally validated the contextual synergy of 2 pathways by demonstrating that a node-specific inhibition of the PI3K-mTOR pathway by GDC-0980 in the presence of carboplatin resulted in (1) an enhanced impairment of DSB repair and (2) a subsequent sensitization to PARPi (i). This effect occurred simultaneously with the inhibition of classic PI3K-mTOR survival signal(s) which induced a robust antiproliferative/proapoptotic effect even in BRCA-competent TNBC cells. The absence of PTEN, on the other hand, sensitized TNBC cells to PARPi in the presence of carboplatin, an effect more pronounced in BRCA-loss. Conclusion: Our data showed that the PI3K pathway cooperates with the DDR pathway in the breast oncogenesis especially basal-like and TNBC. Citation Format: Dey N, Williams C, Krie A, Klein J, Williams K, Carlson JH, De PK, Leyland-Jones B. A tale of two pathways: Mutations in PI3K pathway in TNBC patients matter for the oncogenic cooperation with DNA damage repair pathway [abstract]. In: Proceedings of the 2017 San Antonio Breast Cancer Symposium; 2017 Dec 5-9; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2018;78(4 Suppl):Abstract nr PD4-15.

The role of PI3K activation in transformation was first brought to light by virtue of its association with the transforming oncoprotein Middle T antigen of the Polyoma virus. Subsequently, the discovery of the PTEN tumor suppressor gene and its characterization as a lipid phosphatase with preference for PI-3,4-P2 and PI-3,4,5-P3 brought therapeutic targeting of the PI3K pathway back to the fore. Following the discovery of PTEN direct oncogenic activation of PI3K was found to occur through gene amplification and through the acquisition of activating point mutations in PIK3CA (the gene encoding the catalytic subunit p110α) in a diverse set of malignancies. Lastly, activated oncogenic receptor tyrosine kinases including HER2 and EGFR mediated their transforming activity, at least in part, through PI3K-dependent signaling. Thus, we can envision at least 3 prevalent cancer-genetic states that may elicit a dependence on PI3K, namely, loss-of-function of PTEN, gain-of-function of PIK3CA and oncogenic mutation in receptor tyrosine kinases. The mTOR kinase is a key regulator of cell growth downstream of PI3K. mTOR activity in cancer states is more specifically deregulated in the absence of the normal function of the Tuberous Sclerosis Complex genes (TSC1 and 2). Similarly mutations in LKB1 and PTEN may elicit dependence on mTOR activity. In addition, mTOR activity is a critical regulator of hypoxia signaling. mTOR inhibitors have shown promising clinical activity in states predicated on these pre-clinical discoveries. These observations/discoveries have motivated and set the stage for the development of therapeutic molecules targeting key pathway components. These include the aformentioned allosteric inhibitors of mTOR already registered as oncology therapeutics, and the more recent entrance of PI3K and AKT inhibitors into the clinic. These events herald the opportunity to answer critical therapy based questions in man. The data emerging from the pre-clinical and clinical development of a series of distinct pathway inhibitors targeting mTOR and PI3K will be discussed with a focus on understanding pathway dependence pre-clinically and the role of relevant biomarker data emerging from the clinic and their potential relationship to efficacious dosing. Citation Information: Mol Cancer Ther 2009;8(12 Suppl):PL02-01.

PI3K pathway in cancer

The PI3K signaling pathway is a complex and tightly regulated network that is critical for many physiologic processes, such as cell growth, proliferation, metabolism, and survival. Aberrant activation of this pathway can occur through mutation of almost any of its major nodes and has been implicated in a number of human diseases, including cancer. The high frequency of mutations in this pathway in multiple types of cancer has led to the development of small-molecule inhibitors of PI3K, several of which are currently in clinical trials. However, several feedback mechanisms either within the PI3K pathway or in compensatory pathways can render tumor cells resistant to therapy. Recently, targeting proteins of the bromodomain and extraterminal (BET) family of epigenetic readers of histone acetylation has been shown to effectively block adaptive signaling response of cancer cells to inhibitors of the PI3K pathway, which at least in some cases can restore sensitivity. BET inhibitors also enforce blockade of the MAPK, JAK/STAT, and ER pathways, suggesting they may be a rational combinatorial partner for divergent oncogenic signals that are subject to homeostatic regulation. Here, we review the PI3K pathway as a target for cancer therapy and discuss the potential use of BET inhibition to enhance the clinical efficacy of PI3K inhibitors. Clin Cancer Res; 22(11); 2605-10. ©2016 AACR.

PTEN (phosphatase and tensin homolog on chromosome 10) is a tumor suppressor whose cellular regulation remains incompletely understood. We identified phosphatidylinositol 3,4,5-trisphosphate RAC exchanger 2a (P-REX2a) as a PTEN-interacting protein. P-REX2a mRNA was more abundant in human cancer cells and significantly increased in tumors with wild-type PTEN that expressed an activated mutant of PIK3CA encoding the p110 subunit of phosphoinositide 3-kinase subunit alpha (PI3Kalpha). P-REX2a inhibited PTEN lipid phosphatase activity and stimulated the PI3K pathway only in the presence of PTEN. P-REX2a stimulated cell growth and cooperated with a PIK3CA mutant to promote growth factor-independent proliferation and transformation. Depletion of P-REX2a reduced amounts of phosphorylated AKT and growth in human cell lines with intact PTEN. Thus, P-REX2a is a component of the PI3K pathway that can antagonize PTEN in cancer cells.